Process for the determination of microbial sensitivity to antimicrobial agents



y 27, 1965 1.. s. GOLDMAN 3,197,384

PROCESS FOR THE DETERMINATION OF MICROBIAL SENSITIVITY TO ANTIMICROBIAL AGENTS Filed Dec. 15, 1960 if [Jaw United States Patent 0 3,197,334 PROCESS FOR THE DETERMINATIQN (BF MICRGBEAL SENSITEVETY T0 ANTIMKCRG- REAL AGENTS Leon S. Goldman, New York, N.Y., assignor of ten percent to Harold C. Herman Filed Dec. 13, 196%, Ser. No. 76,438 3 Claims. (Eli. 195-1933) This application is a continuation-in-part of my application, Serial No. 575,083, filed March 30, 1956, and the invention relates to a process and apparatus for determining microbial sensitivity to antimicrobial agents and, more specificially, to a process and apparatus using redox indicators such as salts of various tetrazolium derivatives for the determination of said sensitivity.

In the prior art, the determination of bacterial sensitivity to antibiotics has been performed in the following manner:

A nutrient liquid is prepared and poured into a petri plate or dish and sterilized. On cooling, the nutrient liquid is converted into a rigid solid gel in the dish. The micro-organisms under consideration, obtained from the lesion or from the blood, sputum, or urine of the patient, are then streaked rapidly across the surface of the sterile agar with a sterile applicator. Small paper discs, previously impregnated with various antibiotic solutions and thereafter dried, are placed rapidly on the surface of the inoculated agar. The unit, including the paper discs, is then placed into an incubator kept at 37 degrees centigrade, for a period of 18 to 24 hours. The organisms reveal themselves, after this period of incubation, as discrete colonies along the streaked portions of the agar, except in those areas where they are sensitive to the antibiotics with which they are in contact. A clear zone of inhibition is noticed adjacent the antibiotic discs to which the organisms are sensitive. Occasionally sterile blood is mixed with the liquified agar, prior to pouring, in order to demonstrate the hemolytic reaction of red blood cells, a procedure that is useful in the identification of certain bacteria.

Utilizing the prior art processes, the plates containing the prepared culture media cannot be stored for long periods of time, even when refrigerated, due to the drying and shrinkage of the gel. Many fastidious microorganisms require a renewed fresh surface for optimum growth and so the gel must be reliquified and resolidified prior to use. Furthermore, the process of streaking plates does not lend itself to a uniform distribution of microorganisms on the surface of the gel. Pour plates give better results due to a better distribution of microorganisms but this procedure is time-consuming and is therefore not generally used. I

The prior art processes, moreover, are complex and therefore impractical as an office procedure. Because of the complexity for the physician, these processes are employed almost exclusively in clinical laboratories by trained technicians.

In addition to the difiiculties of storage, uniform distribution and complexity, a period of 18 to 24 hours generally must elapse before any clearly defined inhibition can be detected. Earlier results are occasionally greatly needed and sometimes may even be a life-saving measure.

The objects of this invention are to provide a process and apparatus for the determination of microbial sensitivity to antimicrobial agents wherein said process can be performed rapidily and simply by untrained personnel and wherein said apparatus can be stored ready for use without refrigeration and with minimum storage space and wherein said determination will result in reliable and meaningful data for sensitivity determinations and wherein said determination will conveniently differentiate between bacteriostasis and bactericidal action.

This invention overcomes the difliculties presented by the prior art and accomplishes the foregoing objects by providing a sterile sheet of filter paper which is impregnated at discrete and predetermined positions with a number of antimicrobial agents. When a sensitivity test is to be performed, the inoculum, containing the micro-' organisms under consideration, is mixed in avial containing a sterile nutrient liquid. The contents of the vial are poured over said treated paper and is incubated. The microbial sensitivity to various antimicrobial agents may be determined after a relatively short period of time.

In my application Serial No. 575,083, now abandoned, there is disclosed such apparatus for the determination of bacterial sensitivity to antimicrobial agents wherein a triphenyltetrazolium chloride and an antibiotic or other 7 agent are bound to a filter pad on small premarked areas by means of such non-toxic resinous materials as shellac orrosin. When the filter pad is wetted with an inoculated broth, the slow but immediate horizontal dilfusion of both the redox indicator and antibiotic commences. The colorless indicator, diffusing at sub-lethal concentrations over an area wider than the diffusing antibiotic, is reduced to the colored formazan in the presence of viable bacteria. This reduction results in the formation'of a solid colored zone around the initial point of diffusion when'sensitivity does not occur. When sensitivity exists, a thin colored ring with a clear center is perceived.

The present application represents my better understanding of the invention disclosed and claimed in my application Serial No. 575,083. The scope of this application is broader than my application Serial No. 575,- 083 and includes the subject matter of the application Serial No. 575,083 as embodiments. The entire disclosure of said application Serial No. 575,083 is incorporated herein as though fully disclosed herein. More particularly, this application discloses my improved understanding of the invention with particular emphasis to utilizing any of the numerous tetrazolium redox indicators instead of just a triphenyltetrazolium chloride, and

to a better understanding of the function ofthe resinous material.

An important feature of this invention relates to the use of a sparingly water-soluble, diffusible, relatively nontoxic, salt, containing the tetrazolium ring in it's structural formula, to detect the presence or absence of sensitivity. These tetrazolium salts are normally practically colorless in their oxidized state, but will become highly colored in the presence of viable microorganisms. In-

cluded in this feature is the use of a triphenyltetrazolium chloride compound in the antimicrobial agent solution. I

Anotherfeature of this invention relates to the use of a resinous material in each of the antibiotic solutions. The resinous material prevents the antibiotic from being washed away from its initial and identifying position when the nutrient liquid is added. The resinous material has both a physical and a chemical binding action.

Another feature of this invention relates to adjusting the concentrations of antimicrobial agents and the tetrazolium salt to provide for a greater spreading of the tetralittle or no problem by virtue of its new chemical and physical properties, its method of employment and by employing a control as part of the test.

Still another feature of this invention pertains to the use of a nutrient liquid containing a non-toxic thickening agent. The thickening agent imparts to the liquid the properties of an agar in the sense that bacteria colonies will maintain their positions on the filter pad.

A further feature of this invention pertains to the provision of two sheets of filter paper, one white or light in color and the other black or darker in color. The white or light background facilitates recognition of the highly colored formazan resulting from the tetrazolium salt and the recognition of the pigments produced by some microorganisms; the black or dark background facilitates recognition of the microbial colonies which are usually white or light in color at an earlier stage of their development.

Further features and advantages of this invention will become apparent to those skilled in the art upon consideration of the drawing wherein:

FIGURE 1 is a top view of the apparatus of this invention with the glass top removed;

FIGURE 2 is a sectional view taken along lines 22 in FIGURE 1; and

FIGURE 3 is a bottom view of the apparatus of this invention.

Referring to FIGURES 1 through 3, a disc of porous paper or filter 2, in contact with another disc or porous paper or filter 3 is placed in a petri plate or dish 1. The filter 2 is dyed black or dark and the filter 3 is white or lightly colored. By way of example, the dish 1 may be about 100 millimeters in diameter and about millimeters in height and the filters 2 and 3 slightly smaller in diameter to just cover the bottom of the dish 1. The dish 1 has a cover or lid 5 which substantially seals the dish opening. With the dish 1 covered by the lid 5 to enclose the filters 2 and 3,.the entire unit is sterilized. A

number of antimicrobial solutions, to which have been added certain tetrazolium salts, are then placed on this paper in the previously marked areas 4 and are permitted to dry. As examples of the antimicrobial agents that may be employed are the naturally occurring antibiotics like penicillin, terramycin, erythromycin, etc., the synthetic antimetabolites like the numerous sulfa drugs, or the antiseptic or disinfectants like benzalkonium chlorides, mercurial compounds, phenolics, etc. In FIGURE 1, by way of example, P designates penicillin, Au designates aureomycin, S designates streptomycin, and G designates Gantrisin.

A nutrient broth or liquid is prepared and measured into a small vial, not shown, containing two small glass heads, the vial and its contents all being sterilized. The

volume of the liquid depends upon the size and porosity of the filter pads and should be just enough to completely staurate the pads without leaving excess fluid to flow along the surface of the black. paper 2 when dish 1 is handled. The container. employed in this invention is not limited to a glass petri dish. Disposable plastic dishes ortransparent plastic sacksserve equally well and are even more desirable because they are less expensive.

When a sensitivity test is to be performed, the operator places approximately 0.1 to 0.2 ml. of the inoculum under consideration into the vial containing the sterile nutrient liquid and mixes by shaking, the glass beads aiding in the dispersion of the microbes. The inoculum may be obtained directly from the lesion or from a previously prepared streak or pour plate. The contents of the vial are then poured onto the surface of the black paper 2 and the operation is complete. The dish 1, covered by the lid 5 is placed into a plastic envelope to minimize evaporation and the whole unit is then placed into an incubator. Early sensitivity results are generally obtained in about 4 to 5 hours and confirmatory results are obtained in about 18 to 24 hours. The whole testing unit, representing the prepared dry sterile papers in the sterile glass or colored formazan.

plastic dishes or envelopes plus the sterile nutrient broth may be prepared in quantity long before the test is performed and may be stored at room temperature for long periods of time without deterioration and with minimum storage space.

As the nutrient liquid, one may select any of the standard broths now available in the bacteriology laboratory, or any suitable modification thereof. For example, one may employ heart infusion broth, brain heart infusion broth, tryptose broth, etc. I have found it advantageous to incorporate a thickening agent like methyl cellulose, of viscosity 400i) cps. and at an optimum concentration of 0.8 percent. Other useful thickening agents are gelatin and agar, both at a concentration sufiicient for the solidification of the contents of the vial at room temperature.

The methyl cellulose is particularly useful as a thickening agent, because unlike gelatin and agar, the viscosity of the solution increases with temperature, so that at the incubation temperature of 37 degrees C. the nutrient liquid will be fairly viscous and will impart to the black paper 2 the desired shiny smooth surface without fiowing back and forth when the dish is moved. In this manner, the colonies remain fixedon the shiny surface and do not flow back and forth to obscure the confirmatory results. A selection of 4 or 5 different nutrient liquids will provide for the growth of practically all the pathogenic microorganisms normally encountered.

The antimicrobial solutions are prepared by dissolving the ry materials, in the proper concentrations, in any suitable solvent that will also dissolve the tetrazolium salt. The most useful solvent is methyl alcohol. Occasionally one must use another solvent such as dioxane, N,N-dimethyl formamide, etc., or combinations thereof. The solution of antimicrobial agent and tetrazolium salt is transferred to the premarked areas of the black paper by means of a micropipette or capillary tube and a volume of liquid is deposited over a small area. A residue of antimicrobial agent, comparable to quantities now employed in the agar diffusion method, is deposited on the premarked area of the paper as the solvent evaporates. The nature of the tetrazolium salt, a most important feature of this invention, is hereinafter described.

The function of the black or dark paper 2 is to allow the colonies, most of which are white or light in color, to be readily perceptible. .The function of thewhite or light paper 3 is to reveal the ditfusible pigments produced by some microorganisms. An even more important function of the white paper. isto reveal the reduction of the colorless or almost colorless tetrazolium salt to the highly Intense color changes develop long before any visible colonies are produced. In this manner, one obtains valuable information as to microbial sensitivity in about 4 to 5 hours (sometimes even sooner) with confirmatory results on the black surface overnight. One may simplify the apparatus by omitting the black paper and the thickening agent if only the colorimetric changes are desired.

A typical result in about 4 to 5 hours is'the formation of a colored circle about the antimicrobial agent on the whitepaper 3 when the antimicrobial agent exhibits to inhibition of growth.

When the microorganisms are sensitive to the antimicrobial agent under consideration, the center of the colored area will remain colorless, except for a rather thin colored border. In 18 t024 hours, discrete colonies will be uniformly distributed on the black surface 2 with clear zones of inhibition around the antimicrobial agents to which the organisms are sensitive.

For the purposes of better clarification, the results may be thus summarized:

No sensitivity-The appearance of a wide colored area, about 10 mm. in diameter, and the center of which is also colored, on the White paper directly below the positioned and premarked antiseptics. This solid colored or almost solid area may be perceived after only 4 to 5 hours. In

18 to 24 hours, bacterial colonies may be seen on the black paper with no zone of inhibition around the positioned and marked antimicrobial agents.

Sensizivity.The appearance of a colorless area, surrounded by a thin colored border, about in diameter, on the white paper 3 di ectly below the antimicrobial agent. if such a colorless area appears in about 4 to 5 hours and then the colorless area becomes colored several hours thereafter, or perhaps overnight, this is interpreted to mean that the antimicrobial agent is bacteriostatic and not bactericidal. The initially high concen ation of antibiotichas temporarily arrested the growth of the organism; when the concentration of the antibiotic became sufficiently lower after a time interval, the organism resumed its growth.

A typical result after 4 to 5 hours is illustrated in FlG- URE 3 where 8 is an area of solid colored pigment, about 10 mm. in diameter. The solid area, the color depending upon the type of tetrazolium salt chosen, is indicative of the fact that the growing microorganisms are not sensitive to the antimicrobial agent that has been originally placed directly above this colored area 8 on black paper 2. The colored area is due to the fact that the colorless, or almost colorless tetrazoliurn salt employed in this invention, is enzymatically reduced to a colored formazan by the viable microorganisms. When the organisms are sensitive to a specific agent, a clear area 7 will appear on the white paper 3 surrounded by a thin colored border 6, which has a diameter or" approximately 10 mm. The area 7 is colorless due to the fact that the organisms around the agent to which they are sensitive have been killed by said agent and are therefore not able to reduce the redox indicator to its colored form. A colored border 6 around the colorless area 7 is formed because as the antibiotic diffuses away from its initial position into the surrounding area it rapidly decreases in concentration to the extent that it is no longer effective in inactivating the organism; and since the tetrazolium indicator, present at a much higher concentration and difiuses over a wider area, is reduced to its colored form, the formazan will appear in the form of a colored ring 6.

A feature of this invention relates to the use of a control. The control, not shown in the drawing, is a premarked area C (for control) containing the dry tetrazoliurn indicator Without any antimicrobial agent, said indicator being present at the same amount present on the other premarked areas of the paper. The purpose of the control is to eliminate any possibility of false positive sensitivity results should the tetrazolium salt be partially inhibitory to the organism. Rarely, if ever, does the area under position marked C not turn to a solid colored area. If the control turns to a solid color, the results for the various antimicrobial agents are then examined for toxicity, no toxicity, or bacteriosiasis, asdiscussed above. Occasionally, though rarely, one may note a clear zone with a colored ring about the control. This means that the indicator itself is toxic to the microorganisms. In

practically every case this clear zone will fill almost imsorbed by both papers 2 and 3. Special anaerobic media,

like thioglycollate broth, may also be employed. The pliable plastic sack, as described above, in intimate contact with papers 2 and 3, will help produce anaerobic conditions. One may also increase the concentration of thickening agent to increase the degree of anaerobiosis.

' For best results, papers 2 and 3 should be in intimate contact with each other and, in addition, white paper 3 should be in intimate contact with the bottom of dish 1. This, of course, is unimportant if only the colorimetric findings are desired, as mentioned above, for then the black filter pad and thickening agents are omitted in the apparatus. To insure any liquid'from draining away from any raised portions of the black filter pad, one may cement black paper 2 to white paper 3, and white paper 3 is cemented to the bottom of the dish 1 with a non-toxic hydrophilic cementing agent like purified guar gum. In another embodiment, a white sheet of filter paper is sprayed with a black paint, illustratively of the non-glossy flat type, in order to. coat but one side of said white paper. 1 The sprayed paper is then washed well in boiling Water containing a nonionic detergent like Tween-80 to remove any toxic materials from the sprayed surface and dried. The treated paper hecomes, in effect, functionally equivalent to two different cemented papers, 2 and 3. v

Beforeproceeding with a description of the nature of the tetrazolium salts as employed in the present invention, a brief consideration of some of the background theory and experimental facts is first provided. The toxicity of the various tetrazolium chlorides is presently not well understood. Some authorities are of the opinion that these salts sidetrack the capture of electrons somewhere along the enzymatic redox processes of the microorganism; others are of the opinion that they may behave as uncoupling agents in 'oxidative phosphorylation. In

either event, the various tetrazolium chlorides, of which hundreds have been prepared and the number of-possibilities of new chlorides being virtually unlimited, are all toxic to many microorganisms, as has been stated previously. The toxicity does not appear to be related to he structure of the tetrazolium chloride, its solubility in water, its redox potential, the number of tetrazolium rings contained in the molecule, the final color of the reduced forrnazan, etc.

It may be reasonable to assume that the tetrazolium ion, a cation of fairly high molecular weight, has an atfinity for the negatively charged surface of the microorganism. In other words, I postulate that the toxicity of the tetrazolium ion may be analogous to the toxicity of the cationic surfactants. The tetrazolium ion may affix itself chemically to the cell wall of the microorganism or it may diffuse into the cell itself and exhibit its lethal action. The following observed facts have lead me to the above conclusion:

(1) An organism, such as Staph. aureus is very sensitive to the tetrazolium chlorides. When a pour plate of this organism is prepared, a pronounced zone-of-inhibition will appear about a small disc of paper impregnated with. triphenyltetrazolium chloride placed on said'pour plate. This wide zone of inhibition is colorless. Ittherefore logically follows that reduction and toxicity are not necessarily correlated and that this substance may function as a powerful inhibitor of microbialgrowth without functioning as a redox indicator.

(2) The above-mentioned wide zone of inhibition may be reduced in size by adding a commercially available material, called neutralizing buffer, to the pour plate or disc. Neutralizing buffer (Difco Labs.) is employed when one desires to nullify the toxic effects of cationic detergents in the isolation of microorganisms from restaurant dishes, containers or utensils.

(3) In my .co-pending application Serial No. 14,572 filed on March 14, 1960, now Patent 3,043,751, a series of extremely insoluble tetrazolium salts are described,

having the novel property of being non-toxic to microorganisms. In the preparation of these salts, the tetrazolium nucleus is not changed; the ability of the tetrazolium ring .to open on reduction to form the respective formazan still exists. However, by virtue of its great insolubility and its inability to diffuse, said representative salt will be readily reduced by viable microorganisms without affecting their growth. 7

7 r r r (4) In my parent application Serial No. 575,083, filed on March 30, 1956, now abandoned, a resinous material like shellac or rosin is employed to bind a triphenyltetrazolium chloride to the filter pad. The resinous material, being essentially water-insoluble, will prevent the indicator and antibiotic from being washed away from their initial starting positions when the filter pad is flooded with the inoculated broth and will provide for the slow but immediate release of indicator and antibiotic. With my better understanding of this invention, it appears that the rosin or shellac appear to serve another purpose which is also important. Shellac and rosin contain organic acids of high molecular weight, sparingly soluble in water, said acids forming salts with the triphenyltetrazoliurn ion. In other words, it appears that the active redox indicator in said application is a triphenyltetrazolium salt of the various acidic components of the shellac and rosin. As a matter of fact, one may actually demonstrate the existence of this type of salt in the following manner: a 0.5% solution of shellac is prepared in methyl alcohol and to this solution one then adds powdered triphenyltetrazolium chloride until the concentration of the latter is also 0.5%. To the alcoholic solution of the two components one then adds an equal volume of Water, whereupon a milky turbidity is produced. When this precipitate is isolated by 'centrifugation and washed several times with 50% methyl alcohol (or is dissolved in pure methyl alcohol and reprecipitated by the addition of water) said precipitate exhibits the property of a tetrazolium indicator by turning red in the presence of reducing agents or viable microorganisms. If one bears in mind that triphenyltetra' zolium is soluble in both water and/or methyl alcohol and that shellac is soluble in pure methyl alcohol but insoluble in water, one can explain the presence of the tetrazolium moiety in the milky precipitate only by the conclusion that an insoluble salt of the triphenyltetrazolium ion was produced.

It appears, therefore, that the function of the resinous material in my former application was that of a binding agent, not only by its physical properties of being insoluble and water-repellant, but also by its chemical action, namely that of forming a sparingly soluble new tetrazolium salt, other than the chloride. The present invention is aresult of this improved understanding of the function of the resinous material, and this continuation-in-part application includes as operable illustrations all of the specific compositions and apparatus disclosed and claimed in the parent application Serial No. 575,083. Due to the improved understanding and as hereinafter described, the present invention is considerably broadened.

The present application, accordingly, discloses my better understanding of the invention, with particular emphasis to:

A. A better understanding of the function of the resinous material which appears to be more than merely a binding agent; and

B. The fact that this invention is not limited to a triphenyltetrazolium chloride, since any of the numerous tetrazolium redox indicators may be employed.

(5) In my copending application Serial No. 14,572, filed on March 14, 1960, now patent 3,043,751, I referred to a group of tetrazolium salts with alkaloidal reagents like sulfosalicylic acid, etc., as being toxic. These salts, produced by the anionic precipitants other than the poly acids, are indeed toxic to microbial life when employed only as described in the specification of said application. By saturating a filter pad throughout with said salts, or by suspending said salts in a nutrient liquid (which are discussed in the specification of this pending application), said salts are indeed toxic to microorganisms; but by employing these salts in a manner analogous to the apparatus described in my application Serial No. 575,083, namely to allow them to diffuse horizontally from a small 8 initial area, I have found that these salts are far less toxic than their parent chlorides. This means that greater quantities may be utilized in this manner as compared to the parent tetrazolium chloride bound to the paper by an inert water-insoluble material (like collodion) thus providing for earlier and more striking results.

In other words, the tetrazolium salts formed by combining a tetrazolium ion with an alkaloidal reagent other than the poly acids will result in a salt that is far less toxic than the chloride salt, once again because, in all probability, the concentration of free unbound tetrazolium ion is low indeed to combine with or enter the cell wall of the microorganism. As a matter of fact, it is the utilization of these salts with the precautionary measures of the employment of the control that is a very important feature of the present invention.

(6) Still another bit of experimental evidence to support my hypothesis is the following. When rosin is utilized to bind a triphenyltetrazolium, as disclosed in my application SerialNo. 575,083, said rosin can be shown to bind more triphenyltetrazolium if the treated rosin is first treated with ammonia water and the treated rosin completely evaporated at about 70 C. Both the treated and untreated resins are water-insoluble, but the rosin which has been treated with ammonia has had its abietic acid anhydrides (which appear to have no affinity for the positive tetrazolium ion) converted to the free acid (probably in the form of the ammonium salt), said acid now being able to bind more tetrazolium ions chemically.

The tetrazolium salts, as employed in this invention, are composed essentially of a positively charged or cationic component and a negatively charged or anionic component. The oppositely charged components appear to dissociate but little in water.

The cationic component The cationic component is a tetrazolium derivative of the general formula:

where R R and R are various substituents on said tetrazolium ring. In general, the various substituents may all be identical, as is the case in triphenyltetrazolium, or may even be all different like p-anisyl, piperonyl and phenyl groups; one of the groups may be a carbohydrate like mannose;. all three may be aromatic like p-biphenyl, phenyl, m-nitrophenyl; one or more of the groups may be aliphatic like the methyl group; one or more of the groups may contain an additional tetrazolium ring, like the ditetrazoliums tetrazolium blue and neotetrazolium; one or more of the substituents may contain halogens like the iodo group or other functional groups like the nitro group, one such material being the commercially available iodonitrotetrazolium chloride (INT).

More specifically, R is generally a radical of a diazotizable amine, usually aryl or its equivalent (like pyridine, pyrazine, etc.); R is usually an aryl group or its equivalent (like those derived from phenyl hydrazine); and R is generally almost any monovalent organic substituent.

The colors formed by the various formazans on reduction of the parent tetrazolium salt vary with the nature of the substituents on said tetrazolium salt. In general, the tetrazolium salts are colorless or almost colorless in their oxidized state. The anionic moiety, described below, may impart a color to the salt prior to reduction. However, the reduced formazan is so intensely colored that the color of the original salt, prior to reduction, is light in comparison to its reduced form; the color in the oxidized form being even lighter since rather minute quantities of materials are utilized. One such example is the picrate salt of triphenyltetrazolium; said salt is light yellow in the oxidized state and is a deep red in the reduced state.

The redox potentials of the various tetrazolium salts utilized in this invention are not identical. Said redox potential depends upon the chemical nature of both the cationic and anionic components. In general, the redox potentials of these salts are very close in value to those of the parent chlorides, one such exception being the tetrazolium chlorides of very low redox potential (like iodonitrotetrazolium chloride). In the latter case, the redox potential of the chloride appears to be appreciably increased when said chloride is converted to a new salt of the type disclosed in the present invention. This phenomenon is advantageous, for it now becomes possible to increase the redox potential of an indicator like iodonitrotetrazolium chloride, in a form other than the chloride, for bacterial sensitivity determinations. The redox potential of said chloride is too low, since iodonitrotetrazolium chloride is reduced too rapidly by microorganisms, even in the presence of inhibitory antibiotics.

The chemical nature of the various substituents on the tetrazolium ring appear to have practically little or no efiect on the efiicacy of the redox indicator as employed in the various inventions. I have employed numerous salts with various substituents, as described above, and have found little or no difference in the choice of the cationic component. The cationic components were either obtained from the commercially available chloride, or was synthesized in my laboratory by first producing the formazan; said formazan being oxidized in the conventional manner to the colorless tetrazolium chloride and was then its conversion to another salt as described below. There appears to be no limit to the number of possibilities of cationic tetrazolium component that are operable in this invention.

The anionic component In general, the anionic component is derived from an acid, or negative ion, which will form a sparingly soluble salt other than the chloride when said negative ion is added to a solution of a tetrazolium chloride in the presence of water. I have successfully employed such materials as picric acid, sulfosalicylic acid, trichloroacetic acid, ferrocyanic acid, ferricyanic acid, nitroprussic acid, the dichromate ion, the persulfate ion, etc. Many of the anionic precipitants are known as alkaloidal reagents and are used in the laboratory in the detection and extraction of-proteins. Alkaloida-l reagents of the type disclosed in my patent application Serial No. 14,572., now Patent 3,043,751, are not suitable in the present invention because these salts are so insoluble thatthey exhibit practically no diffusion across a wet filter pad. Other alkaloidal reagents of high molecular Weight, not claimed in said pending application, may be utilized in the present invention, particularly when a tetrazolium salt is prepared by adding a poly acid of low concentration to a tetrazolium chloride of high concentration and generally by not strongly acidifying the mixture. 7

It is interesting to note that the nitrate ion, an anion of simple structure, will render most proteins sparingly soluble at their iso-electric points and so one may term the nitrate as "a protein precipitant. The nitrate salt of the tetrazoliums may be successfully employed as the redox indicator of the present invention.

The reagents employed to prepare said new tetrazolium salts are not necessarily restricted to the protein precipitants. Certain colloidal materials, negatively charged or having acidic functional groups as an integral part of said colloid, have the property of forming salts with the positive tetrazolium ions having the desired characteristics, as recited below. Such negatively charged particles may be derived from gum mastic, gum arabic,

the acidic fractions of shellac or rosin, etc.; said mat rials being aggregates of high molecular weight. One may also include such colloids as the proteins at a pH at or above their iso-electric points, for-at these pH values the proteins have the free negative carboxylate groups to combine with the positive tetrazolium ion. Examples of three such proteins successfully employed in this invention are casein, globulin and globin. The salts produced by the combination of the negative colloids with the positive tetrazolium ions are not sharply defined chemical compounds and are amorphous solids, unlike a salt like triphenyltetrazolium sulfosalicylate which is a true salt that can be easily crystallized from methyl alcohol and water. The ill-defined chemical nature of said colloidal salts, which do not form from stoichiometric proportions, may be due to the considerable adsorption that occurs on the surface of these colloids. The illdefined chemical nature of said colloidal salts may also explain why a wide range of concentrations of the tetrazolium salt and the resin are operative. A considerable excess of either triphenyltetrazolium chloride or of sh llac or rosin still is operable.

The new redox indicators, as employed in this invention, are thus prepared by combining a cation containing the tetrazolium ring as part of its chemical structure with an anionic precipitant like the alkaloidal reagents, acidic colloids like certain gums, resins and proteins, to form a new composition of matter for the determination of microbial sensitivity to antimicrobial agents when employed in the above-described apparatus. The: new redox indicators should have the following properties, for optimum results: I

1) Reduced solubility in water.-The salts are generally sparingly soluble in water, the new salt of any one tetrazolium ion generally being less soluble in water than the parent tetrazolium chloride.

(2) Reduced t0xiciiy.It is conceded that the variou new tetrazolium salts of this invention are toxic to microbial life. They are, however, far less toxic than their parent chlorides as determined by the serial dilution methods. It must be emphasized that in this invention the redox indicators are utilized in a novel manner, namely,

they are permitted to ditiuse horizontally across a wet filter pad from an initial point of small area. Under these conditions said salts are released at sub-lethal'com centrations over'an area wider than the area coveredby the diffusing antibiotics; when microorganisms are inhibited by the indicator (in unusualcircumstances) one refers to the control for the proper interpretation, as has been mentioned above. When utilized as described in this invention, said new tetrazolium salts can be con- Sidered to be nontoxic, for all practical purposes.

(3) Low degree of i0nizati0n.The new salts appear to ionize but little in water, as evidenced by their low electrical conductivity. Additional evidence of their low degree of ionization may be the fact that when a picrate ion, a ferricyanide ion, a ferrocyanide ion, a dichromate ion (all highly colored ions in their free ionic state in water), is combined with one of the tetrazolium ions, said new salts are but slightly colored when wetted. This indicates that the highly colored ions are essentially bound in the salt in an un-ionized state as an inner salt.

(4) Difiusibility.The new redox indicators of this invention are able to diffuse across a wet filter pad, said diffusion being illustratively at least twice the area of the sensitivity determinations is approximately 240% in a non-aqueous medium. Said solution of newsalt, also containing a suitable concentration of antimicrobial agent as mentioned above, is added to the pro-marked area of ll the filter pad by means of a micropipette or capillary tube. The concentration range of 210% is merely a convenient range and is illustrative. The concentration of redox indicator employed in the manufacture of this equipment is actually unimportant since it is the residue remaining in the small'premarked areas of the filter pad upon evaporation that is operative. Furthermore, the actual quantity of the redox indicator residue also appears to be not critical in view of the fact that said redox indicator is deposited in a small pre-marked area as a sparingly soluble salt. Diffusion from said pre-marked area will begin almost immediately after the filter pad is wetted. However, the concentration of the diffusing salt across any chosen boundary is primarily determined by the intrinsic solubility of the salt, said concentration beginning essentially as that of a saturated solution. Since the various redox indicators employed in this invention are all, sparingly soluble in Water and are in about the same order of solubility, it appears that the actual quantity deposited on said premarked areas will not materially affect the efiicacy of the diagnostic test as long as sufficient material is deposited to diffuse over an area wider than the ditfusion of the antibiotic. It should also be noted that the thickness and porosity of the filter pad will also be one of the factors in determining the quantity of redox indicator that is employed.

Another function of the sparingly-soluble redox indicator, as employed in this invention, is the property of said salt to prevent the water-soluble antimicrobial agents, deposited along with the redox indicator on the same premarked areas of the filter pad, from being washed away when the inoculated nutrient broth is poured on the filter pad. The redox indicator accomplishes this task by virtue of its relative insolubility in water; the pre-marked areas are not immediately wetted when the test is performed and in this respect the relatively insolublesalt, present at a much higher quantity than the antibiotic, behaves in essence as a binding agent for the antibiotic. While an additional binding .agent is theoretically and practically unnecessary in this invention, one may add an additional binding agent to the solution of tetrazolium salt and antibiotic before depositing this solution on the pre-marked areas of the filter pad as an additional safeguard. Any water-insoluble non-toxic material that will provide for an almost immediate diffusion of redox salt and antimicrobial agent (except for a very brief lag period) may be employed. Examples of suitable material are collodion, shellac, rosin, silicone resins, hydrocarbons of high molecular weight, etc.

The tetrazolium salts of this invention are conveniently prepared by means of a double-decomposition reaction. The starting material may be a solution of a highly ionized tetrazolium salt, such as the chloride, to which is then added a solution of an anionic precipitant of the types described above. If both reactants are Water-soluble (as are, for example, triphenyltetrazolium chloride and sulfosalicylic acid), the reaction is carried out by simply mixing the aqueous solutions of the two reactants at approximately equivalent concentrations. The precipitate formed is then best purified by repeatedly washing with water and centrifugation, finally crystallizing the new salt from a suitable solvent mixture, if feasible (triphenyltetrazolium sulfosalicylate may be easily crystallized from a hot 1:1 methyl alcohol-water mixture).

If one of the components is water-soluble and the other is Water-insoluble (as are, for example neotetrazolium chloride and picric acid, the latter being water-soluble), the same procedure is followed except that the .waterinsoluble component is dissolved in a non-aqueous solvent that is miscible with water (like methyl alcohol, ethyl alcohol, acetone, etc.). The solutions of the two reactants are then mixed as above and the precipitated salt purified as above.

If both components are sparingly soluble in Water (like iodonitrotetrazolium chloride and shellac), a non-aqueous agent.

12 solvent that is miscible with water is used to dissolve both components to form a clear solution. Upon diluting the said solution of the two components with an approxi mately equal volume of water, a precipitate will form. This precipitate, representing the crude new salt, is purified by centrifugation, redissolving in pure non-aqueous solvent and diluting with water; this procedure is repeated several times and the new salt is crystallized from an appropriate solvent mixture, if feasible.

The Double Decomposition Method of preparing the redox indicator salts of this invention is merely illustrative, as other convenient methods may also be employed. For example, if the colored formazan is first prepared, as is generally the case when new tetrazolium redox indicators are synthesized, said formazan may be oxidized (and thereby converted to the colorless salt) to the tetrazolium salt desired in the present invention in the presence of an excess of anionic precipitant. The advantage of this method is that the preparation of the chloride salt is eliminated thereby saving a step in the preparation of the new salt. The anionic precipitant should of course be practically unaffected by the oxidizing agent and, better still, the anionic precipitant should be the oxidizing For example, triphenyltetrazolium nitrate may be directly prepared from the red formazan by the oxidation of the latter in excess nitric acid; the ferricyanide salt of various tetrazoliums may be prepared by the oxidation of the corresponding formazan in concentrated potassium ferricyanide; the dichromate salt of a tetrazolium may be conveniently prepared by the oxidation of the corresponding formazan with excess potassium dichromate, etc.

The materials and methods employed in the preparation of the class of redox indicators, as employed in this invention, are therefore many and varied. They are all directed, however, to combining a cation, containing at least one tetrazolium ring, in a highly ionized state, with an anionic precipitant, said combination taking place generally in the presence of water, to form a sparingly water-soluble, poorly dissociated inner salt, said redox indicators being either amorphous salts or crystalline salts; said salts formed from their cationic and anionic components in exact stoichiometric proportions or formed over a fairly wide, range of combining weights due to adsorption phenomena; said salts generally being less toxic to microorganisms and less soluble in water than the chloride salts of the same tetrazolium ion; said salts, despite their relative insolubility in Water, are nevertheless readily difr'usible across a wet filter pad.

Although this invention has been disclosed and illustrated with reference to particular applications, the principles involved are susceptible of numerous other appli cations which will be apparent to persons skilled in the art. The invention is, therefore, to be limited only as indicated by the scope of the appended claims.

I claim:

1. A process for the determination of bacterial sensitivity to antibiotics, comprising the steps of placing a disc of dark filter paper which is utilized for the easier perceptibility of bacterial colonies against a disc of light filter paper which is utilized for the easier perceptibility of pigment and hemolytic process, then impregnating premarked areas on the two discs with various antibiotic solutions to provide for the rapid detection of said bacterial sensitivity, each of the antibiotic solutions having a triphenyltetrazolium chloride to provide for the rapid determination of bacterial sensitivity, a water-insoluble non-toxic resin from the group consisting of rosin and shellac to prevent the antibiotics from being washed away and to provide for a slow ditfusion of the triphenyltetrazolium chloride into the surrounding areas of said papers, and then drying the discsimpregnated by the solutions at the marked areas, said non-toxic resin being added as a. solution of 0.5 percent concentration.

2. A process for the determination of bacterial sensitivity to antibiotics, comprising the steps of inoculating a nutrient liquid with the bacteria under consideration and pouring a premeasured quantity of said inoculated nutrient liquid into a dish containing a disc of filter paper which has been impregnated with various antibiotics, with each of said antibiotics being bound to said paper together with a triphenyltetrazolium chloride by means of a Water-insoluble resinous material from the group consisting of rosin and sh llac, the concentration of the Water-insoluble resinous material being at least 0.5 percent.

3. A process for the determination of bacterial sensitivity to antibiotics, in accordance with claim 2, wherein the antibiotic residues on said paper is about 30 micrograms, said triphenyltetrazolium chloride solution being of a concentration of about 0.5 percent and said resinous material being in a solution between 0.5 percent and 1 percent before they are applied to said papers and evaporated to dryness.

References Cited by the Examiner UNITED STATES PATENTS OTHER REFERENCES Am. Jour. of Clin. Pathology, vol. 23, pages 1168 to Antibiotics and Chemtherapy, vol. 5, No. 7, page 382. Four. of Lab. and Clin. Med, vol. 44, No. 4, pages 589 and 590, 1954.

AQLOUIS MONACELL, Primary Examiner.

ABRAHAM H. WINKELSTEIN, Examiner. 

1. A PROCESS FOR THE DETERMINATION OF BACTERIAL SENSITIVITY TO ANTIBIOTICS, COMPRISING THE STEPS OF PLACING A DISC OF DARK FILTER PAPER WHICH IS UTILIZED FOR TH EASIER PERCEPTIBILITY OF BACTERIAL CONONIES AGAINST A DISC OF LIGHT FILTER PAPER WHICH IS UTILIZED FOR THE EASIER PERCEPTIBILITY OF PIGMONT AND HEMOLYTIC PROCESS, THEN IMPREGNATING PREMARKED ATEAS ON THE TWO DISCS WITH VARIOUS ANTIBIOTIC SOLUTIONS TO PROVIDE FOR THE RAPID DETECTION OF SAID BACTERIAL SENSITIVITY, EACH OF THE ANTIBIOTIC SOLUTIONS HAVING A TRIPHENYLTETRAZOLIUM CHLORIDE TO PROVIDE FOR THE RAPID DETERMINATION OF BACTERIAL SENSITIVITY, A WATER-INSOLUBLE NON-TOXIC RESIN FROM THE GROUP CONSISTING OF ROSIN AND SHELLAC TO PREVENT THE ANTIBIOTICS FROM BEING WASHED AWAY AND TO PROVIDE FOR A SLOW DIFFUSION OF THE TRIPHENYLTETRAZOLIUM CHLORIDE INTO THE SURROUNDING AREAS OF SAID PAPERS, AND THEN DRYING THE DISCS IMPREGNATED BY THE SOLUTIONS AT THE MARKED AREAS, SAID NON-TOXIC RESIN BEING ADDED AS A SOLUTION OF 0.5 PERCENT CONCENTRATION. 